skip to main content

DOE PAGESDOE PAGES

Title: Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even ifmore » their epitopes overlap.« less
Authors:
 [1] ; ORCiD logo [2] ;  [2] ;  [2] ;  [3] ; ORCiD logo [4] ; ORCiD logo [2]
  1. Univ. of Zurich (Switzerland). Dept. of Biochemistry; Genentech, San Francisco, CA (United States). Dept. of Early Discovery Biochemistry
  2. Univ. of Zurich (Switzerland). Dept. of Biochemistry
  3. Univ. of Zurich (Switzerland). Dept. of Biochemistry; Federal Inst. of Technology, Zurich (Switzerland). Dept. of Biosystems Science and Engineering
  4. Univ. of Zurich (Switzerland). Dept. of Biochemistry; SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
Publication Date:
Grant/Contract Number:
AC02-76SF00515; 310030B_166676
Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 7; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Research Org:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org:
USDOE; Schweizerische Nationalfonds
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Fluorescent proteins; Protein design; Protein folding; X-ray crystallography
OSTI Identifier:
1419739

Hansen, Simon, Stüber, Jakob C., Ernst, Patrick, Koch, Alexander, Bojar, Daniel, Batyuk, Alexander, and Plückthun, Andreas. Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity. United States: N. p., Web. doi:10.1038/s41598-017-15711-z.
Hansen, Simon, Stüber, Jakob C., Ernst, Patrick, Koch, Alexander, Bojar, Daniel, Batyuk, Alexander, & Plückthun, Andreas. Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity. United States. doi:10.1038/s41598-017-15711-z.
Hansen, Simon, Stüber, Jakob C., Ernst, Patrick, Koch, Alexander, Bojar, Daniel, Batyuk, Alexander, and Plückthun, Andreas. 2017. "Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity". United States. doi:10.1038/s41598-017-15711-z. https://www.osti.gov/servlets/purl/1419739.
@article{osti_1419739,
title = {Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity},
author = {Hansen, Simon and Stüber, Jakob C. and Ernst, Patrick and Koch, Alexander and Bojar, Daniel and Batyuk, Alexander and Plückthun, Andreas},
abstractNote = {Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.},
doi = {10.1038/s41598-017-15711-z},
journal = {Scientific Reports},
number = 1,
volume = 7,
place = {United States},
year = {2017},
month = {11}
}

Works referenced in this record:

Rapid protein-folding assay using green fluorescent protein
journal, July 1999
  • Waldo, Geoffrey S.; Standish, Blake M.; Berendzen, Joel
  • Nature Biotechnology, Vol. 17, Issue 7, p. 691-695
  • DOI: 10.1038/10904

The Green Fluorescent Protein
journal, June 1998

Engineering and characterization of a superfolder green fluorescent protein
journal, December 2005
  • Pédelacq, Jean-Denis; Cabantous, Stéphanie; Tran, Timothy
  • Nature Biotechnology, Vol. 24, Issue 1, p. 79-88
  • DOI: 10.1038/nbt1172

Evolution of new nonantibody proteins via iterative somatic hypermutation
journal, November 2004
  • Wang, L.; Jackson, W. C.; Steinbach, P. A.
  • Proceedings of the National Academy of Sciences, Vol. 101, Issue 48, p. 16745-16749
  • DOI: 10.1073/pnas.0407752101

Coot model-building tools for molecular graphics
journal, November 2004
  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158